Photodynamics of latex nanospheres examined using two-photon fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is a highly sensitive and selec tive technique with which one can analyze highly localized environments usi ng the fluctuations in the average fluorescence of a probe fluorophore. Two -photon FCS has been proposed as a method to minimize photodamage to the pr obes and living organisms under observation. While multiphoton excitation r educes photodamage outside the focal volume, photodynamic effects within th e volume can be substantial. The dynamics occurring in the focal excitation volume of a two-photon confocal microscope are examined using FCS of dye-l oaded, 100 nm diameter polystyrene spheres. Multiphoton photodynamic events are observed which cannot be explained either by simple local solution hea ting or by intersystem crossing within a single excited dye molecule. The l aser power dependence of photodynamics process suggests that a four-photon absorption by polystyrene and subsequent ablation of the sphere is the mech anism of fluorescence-reducing photodamage.